In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein-free medium: effects of carbohydrates and amino acids

1999 ◽  
Vol 11 (2) ◽  
pp. 127 ◽  
Author(s):  
J. M. Lim ◽  
B. C. Lee ◽  
E. S. Lee ◽  
H. M. Chung ◽  
J. J. Ko ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Fertilization ◽  
In Vitro Maturation ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Pronuclear Formation ◽  
Bovine Oocytes

This study was conducted to examine the effects of carbohydrates and amino acids on the maturation and fertilization of bovine oocytes. To evaluate the effect of each treatment without any unpredictable interference, oocytes were cultured in a simply defined medium (modified Tyrode’s medium; mT) without the addition of hormones and proteins. In Experiment 1, oocyte maturation to the metaphase-II stage was significantly (P<0.0001) enhanced after the addition of glucose (5.6 mМ), lactate (10 mМ) and/or pyruvate (0.5 mМ) to mT (37–74%) than after no addition (0%). In mT supplemented with glucose, the addition of 19 essential and non-essential amino acids (aa; 0, 0.01, 0.1, 1, 5 or 10%) did not further improve in vitro maturation (Experiment 2) or in vitro fertilization (Experiment 3) of oocytes. However, more (P<0.05) pronuclear formation after in vitro-insemination was found in oocytes matured in mT with 1% aa and glucose than in oocytes matured in mT with glucose alone (56% vs. 35%). Penetration of spermatozoa into the ooplasm was initiated at 3 h after insemination and pronuclear formation from 8 h (Experiment 4). When cultured inseminated oocytes were examined up to 192 h post insemination, a significant (P<0.05) increase in the number of 2-cell (18 v. 38%) and 8-cell embryos, (7 v. 20%) and morulae (0 v. 8%) was found after the addition of 1% aa to mT with glucose than after no addition (Experiment 5). A limited number of oocytes matured in mT with aa and glucose developed to the blastocyst stage (6%). These results indicate that exogenous carbohydrates and amino acids are prerequisites for the maturation and fertilization of bovine oocytes in vitro. Glucose alone promotes the nuclear maturation of oocytes, whereas amino acids aid the pronuclear formation of fertilized oocytes.

ТРАНСВАГИНАЛЬНАЯ АСПИРАЦИЯ ФОЛЛИКУЛОВ И СПОСОБНОСТЬ OPU-ООЦИТОВ КОРОВ К ЭМБРИОНАЛЬНОМУ РАЗВИТИЮ В УСЛОВИЯХ IN VITRO

2019 ◽  
pp. 17-19
Author(s):  
G.N. SINGINA ◽  
V. HAVLICEK ◽  
N.P. TARADAYNIK ◽  
R.Y. CHINAROV ◽  
T.E. TARADAYNIK ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Maturation In Vitro ◽  
Transvaginal Aspiration ◽  
Cattle Reproduction ◽  
Ovarian Follicular Growth

Представлены результаты трансвагинальной аспирации ооцитов коров, а также оценен их потенциал к эмбриональному развитию после оплодотворения в условиях in vitro. Донорами яйцеклеток являлись половозрелые телки симментальской породы в возрасте 1619 мес. Животныедоноры (n7) перед проведением процедуры Ovum Pickup (OPU) были гормонально обработаны с целью стимуляции роста фолликулов. Количество выделенных ооцитов от индивидуальных доноров составило в среднем 7,7 ооциткумулюсных комплексов (ОКК), что соответствовало степени извлечения 54,57,7. Доля ОКК хорошего качества, рассчитанная от общего числа извлеченных ОКК, между отдельными животными существенно не различалась (значения варьировали от 60,0 до 75,0) и в среднем составила 67,21,9. ОКК с признаками нормальной морфологии подвергали in vitro процедурам созревания, оплодотворения и последующего культивирования до стадии бластоцисты. Доля раздробившихся ооцитов и выход бластоцист после in vitro осеменения яйцеклеток коров равнялась 75,7 и 24,3, соответственно. В целом от одного донора за сессию OPU было получено 1,3 эмбриона на стадии бластоцисты, содержащих в среднем 89,8 ядра. Оцененный способ экстракорпорального оплодотворения OPUооцитов коров позволяет получать эмбрионы, пригодные для замораживания и трансплантации реципиентам и может быть использован в программах по воспроизводству желаемых генотипов у крупного рогатого скота.In the present work, we report the data on transvaginal aspiration of bovine ovarian follicles and estimation of in vitro embryo development competence of collected oocytes. The oocytes were collected by ovum pickup OPU from seven 1619 monthold Simmental heifers, previously hormonallytreated in order to stimulate ovarian follicular growth. In average, 7.7 oocytecumulus complexes (OCCs) per heifer per OPU session were collected that corresponded to 54.57.7 of recovery rate. Morphologically, 60.075.0 of OCCs were the good quality and this rate did not significantly differ between the animals. Good quality OCCs (total n37) were then subjected to in vitro maturation, in vitro fertilization and in vitro embryo development up to blastocyst stage. Cleavage and blastocyst rates were 75.7 и 24.3 , respectively. In total, 1.3 blastocysts were obtained per cow per OPU session in average these blastocysts contained 89.9 cells. In conclusion, we developed the methodology of in vitro fertilization of bovine OPUcollected oocytes that allowed obtaining the blastocysts potentially suitable for freezing and transplantation to recipients. This approach can be used to multiply desired genotypes in cattle reproduction.

scholarly journals IN VITRO MATURATION OF BOVINE OOCYTES FOR IN VITRO FERTILIZATION

2009 ◽  
Vol 15 (1) ◽  
pp. 16-19
Author(s):  
Ioan GROZA ◽  
Simona CIUPE ◽  
Mihai CENARIU ◽  
Emoke PALL ◽  
Anamaria PETREAN
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
In Vitro Maturation ◽  
Cultivation Conditions ◽  
Vitro Fertilization ◽  
Morphological Evaluation ◽  
17Β Estradiol ◽  
Bovine Oocytes

The objective of the present study was to asses the quality of various cultivation media used for the maturation of bovine oocytes that are prepared for IVF. Upon collection from slaughtered bovine ovaries and after morphological evaluation, a total number of 513 viable oocytes have been selected for cultivation, being divided into 3 batches, 171 oocytes / batch. The oocytes belonging to batch 1 were cultivated in TCM 199 NaHCO3 + 10% FCS + FSH 20 μl/ml. The oocytes belonging to batch 2 were cultivated in TCM 199 NaHCO3 + 10% FCS + HCG 2.3 x 103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml. The oocytes belonging to batch 3 were cultivated in TCM 199 NaHCO3 + 10% FCS + 17β estradiol 1 μl/ml + FSH 20 μl/ml. The cultivation conditions, for all three batches, were: 24 hours at 39°C, 5% CO2. Spermatozoa have been prepared using the Percoll method and IVF of the matured oocytes has been performed. Embryonic development has been assessed 72 hours and then up to 10 days after IVF. The results showed the superior quality of the oocytes belonging to batch 2 and matured using TCM 199 NaHCO3 + 10% FCS + HCG 2.3x103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml, as their use for IVF yielded the highest number of viable embryos.

scholarly journals Oxygen tension and oocyte density during in vitro maturation affect the in vitro fertilization of bovine oocytes

2015 ◽  
Vol 36 (6Supl2) ◽  
pp. 4277
Author(s):  
Angelo Bertani Giotto ◽  
Daniela Dos Santos Brum ◽  
Francielli Weber Santos ◽  
Antonio Carlos Galarça Guimarães ◽  
Cibele Garcia Moreira Gonçalves ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
In Vitro Fertilization ◽  
Oxygen Tension ◽  
In Vitro Maturation ◽  
Ros Production ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation ◽  
Bovine Oocytes

Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20%) with different oocyte densities (1:10?l or 1:20?l) in the in vitro maturation (IVM) of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH) and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P>0.05). In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P<0.05). Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P<0.05). In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P<0.05). Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte density (P<0.05). In conclusion, the results of this study indicate an interaction between oxygen tension and oocyte density, which increases ROS production in certain associations and subsequently influences the rates of in vitro fertilization of bovine oocytes. The improved rates of IVF were obtained when IVM was conducted using 20% oxygen tension and high oocyte density (1:20 ul).

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